General About the Technical Field of the Invention
The invention relates to a process for the separation of a substance by adsorption to non-packed beds containing beads which exhibit structures (ligands) with affinity to the substance. Non-packed beds may be generated by expanding/fluidizing sedimented beads by an upward or a downward flow of fluid, the direction of flow being depending on the density of the beads used. A liquid sample containing the substance to be adsorbed is introduced into the flow after expansion. A less effective non-packed bed is generated by agitating suspendible beads with the aid of a turbulent flow or by mechanical stirring.
By selecting a bead population which includes beads varying in sizes and/or densities and using this population in an expanded bed, it is possible to obtain a so-called classified bed in which larger beads and beads with higher densities are located below smaller beads and beads with lower densities. The backmixing in these type of beds becomes low and they have been named stable expanded beds (sometimes stable fluidized beds). An alternative way of stabilizing a fluidized bed is by incorporating magnetic filler particles into the beads and apply a magnetic field during the fluidization. Stabilization by a magnetic field is an example that stable expanded beds can be achieved without using beads covering a certain size/density range. The adsorption in stable fluidized beds will take place during plug flow as in a chromatographic process in a packed bed. The number of theoretical plates will be high. In case the non-packed bed is generated by a turbulent flow or by agitation, backmixing will be high and the adsorption will take place in a batch-wise mode. For a short, recent survey of the field, see the introductory part of Thommes et al., Biotechnol. Bioengin. 48 (1995) 367-374.
Back-mixing in a bed is often measured as axial dispersion (xe2x80x9cvessel dispersion numberxe2x80x9d), see Levenspiel, xe2x80x9cChemical Reaction Engineeringxe2x80x9d 2nd Edition, John Wiley and Sons (1972). For stable expanded beds, the vessel dispersion number will preferably be  less than 75xc3x9710xe2x88x923, more preferably  less than 20xc3x9710xe2x88x923, which corresponds  greater than 5, more preferably  greater than 30 theoretical plates. For total back-mixing, the number of plates will be 1.
Expansion/fluidization of the bed is normally effected in a column having provided at each of its ends a net structure covering the cross-sectional area of the column, or some other perforated device which will not generate turbulence in the flow. See, for instance, WO-A-9218237 (Pharmacia Biotech AB, Uppsala, Sweden) . The similar effect has also been claimed for a system utilizing a stirred inlet flow (WO-A-9200799; Kem-En-Tek/Upfront Chromatography A/S). Also other distributors are likely to be feasible.
Subsequent to adsorption, elution can be effected directly from the expanded bed. Alternatively, the bed may be allowed to settle and adsorbed material eluted from the bed with the aid of a fluid flow often introduced in a direction opposite to that in which the bed was expanded.
The fluid is often aqueous (for instance buffers dissolved in water), but also other liquids may be used.
Pharmacia Biotech AB (Uppsala, Sweden) markets Streamline(copyright) which in its first version utilized porous beads of agarose with quartz particles as filler material (WO-A-9218237, Pharmacia Biotech AB). In a later version, the quartz particles were replaced with fillers of higher densities than quartz (PCT/SE96/01431, filed with priority from Nov. 7, 1995, Pharmacia Biotech AB). Another main supplier is Bioprocessing Ltd. (Durham, England) whose porous glass beads (Prosep(copyright)) can be used for chromatography on expanded beds (Beyzavi et al, Genetic Engineering News, Mar. 1, 1994 17)). Still another supplier is Sepracor.
U.S. Pat. No. 4,976,865 (Sanchez, et al, CNRS) teaches fluidized beds and the use of segmented columns to mimic the multi-step adsorption taking place in packed as well as stabilized expanded beds. The beads used in the experimental part are glass particles (Spherosil) that have been coated.
WO-A-9200799 (Kem-En-Tek; Upfront Chromatography) discloses a large number of fillers and polymeric materials that can be combined to produce beads intended for adsorption in fluidized beds. Each bead contains two or more filler particles.
WO-A-8603136 (Graves and Burns; University Patents Inc) discloses beads containing magnetic filler particles and their use in fluidized beds stabilized by an externally applied magnetic field. See also Burns et al., Biotechnol. Bioengin. 27 (1985) 137-145; and Lochmxc3xcller et al., J. Chem. Tech. Biotechnol. 40 (1987) 33-40.
In chromatography on packed beds it has earlier been suggested to use porous beads, the pores of which wholly or partly have been filled with hydrophilic gels carrying affinity ligands, such as ion exchange groups. One example is Macrosob-K which is macroporous kieselguhr which has been filled with agarose which in turn has been derivatized to exhibit DEAE or CM ion exchange groups (Macrosorb-KAX.DEAE and Macrosorb-KAX.CM, respectively (GB-A-1,586,364, Miles). This latter type of materials have also been applied in fluidised bed chromatography (Bite et al., In: Verrall et al., Separations for Biotechnology (1987), Elles Horwood Ltd, Chapter 13, 193-199.
Lochmxc3xcller et al., Sep. Sci. Techn. 22(11) (1987) 2111-2125 discloses a comparative study of adsorptions on a packed bed, a fluidized bed in the quiescent state and a magnetically stabilized bed. The beads used are made of Amberlite XAD, which have been coated adsorptively with a synthetic polymer whereafter an affinity ligand for the substance to be adsorbed has been linked to one terminal end of the polymer. Magnetic particles are present in the coat.
Problems Related to Earlier Fluidized Bed System
In the case of adsorption processes on expanded and/or fluidized beds, there is an expressed desire to have the highest possible productivity. Important variables that should be taken into acoount to achieve this is to use beads with the highest possible breakthrough capacity for the substance to be adsorbed and also to increase the flow rate. However, increasing the flow rate leads to decreased breakthrough capacity and also an increased risk for elutriation of beads. One way of solving the elutriation problem is to increase the density of the beads by including filler materials in them. However, filler material as a rule will have a detrimental effect on breakthrough capacity, the size of this effect being dependent on various factors such as the flow rate, the pore size of the beads, the structure that binds the substance to be adsorbed, the substance to be adsorbed etc.
One way of increasing the breakthrough capacity for filler matrices in bead form has recently been presented in International Patent Application PCT/SE96/01431 (Pharmacia Biotech AB), the content of which is hereby incorporated by reference. See below.
A first objective is to improve total yields in adsorption processes on fluidized beds.
A second objective is to improve productivity for adsorption processes on fluidized beds.
A third objective is to provide filler matrices that have improved breakthrough capacity in fluidized beds.
We have now realized that the above-mentioned problems can be solved by utilizing beads in which the affinity structure/ligand is linked to the base matrix of the beads via an extender.
The positive effect caused by an extender is believed to reside in the fact that it will provide the inner surfaces (pore surfaces) and/or outer surfaces of the beads with a flexible polymer layer that is permeable to macromolecules and other molecules allowed to pass the bed. This will cause an increase in the effective interacting volume as well as in the steric availability of the affinity structures/ligands for the substance to be adsorbed. This in turn will increase the mass transfer rate as well as the total capacity available.
Accordingly the first aspect of the invention is an separation method on a fluidized bed or on a stirred suspension as described above. The characteristic feature is that the affinity structure utilised is attached to the base matrix via an extender. Preferentially the attachment is covalent, meaning that in the preferred mode there is no non-covalent link between the affinity structure and the base matrix.
The Extender
Suitable extenders should be hydrophilic and contain a plurality of groups selected, for instance, among hydroxy, carboxy, amino, repetitive ethylene oxide (xe2x80x94CH2CH2Oxe2x80x94), amido etc. The extender may be in the form of a polymer such as a homo- or a copolymer. Hydrophilic polymeric extenders may be of synthetic origin, i.e. with a synthetic skeleton, or of biological origin, i.e. a biopolymer with a naturally occurring skeleton. Typical synthetic polymers are polyvinyl alcohols, polyacryl- and polymethacrylamides, polyvinyl ethers etc. Typical biopolymers are polysaccharides, such as starch, cellulose, dextran, agarose. The preferred polymeric extenders are often water-soluble in their free state, i.e. when they are not attached to the base matrix. Water-insoluble polymers or polymers of low original hydrophilicity may have been rendered more hydrophilic by introducing hydrophilic groups on them. In particular so called polyhydroxy polymers and other polymers lacking polypeptide structure are believed to be preferred.
The length (size) of the optimal extender will depend on several factors, such as number of attachment points to the base matrix of the beads, type of extender, the structure and size of the affinity ligand as well as the number thereof per extender molecule, crosslinking degree etc. The flexibility of the layer formed by the extender will be increased for a decrease in the number of attachment points to the base matrix per extender molecule and/or in the degree of crosslinking of the extender. Extenders enabling one-point attachment, for instance at a terminal monomeric unit, may be small. For polymeric extenders for which attachment and/or crosslinking is possible at several monomeric units, it is believed that larger extenders are preferred. For the above-mentioned type of polymers, for instance, it is believed that the most suitable polymers should contain at least 30 monomeric units, which for polysaccharides like dextran indicates a Mw greater than 5000 Da.
In order to control the flexibility of the extender, it is often advantageous to first activate the base matrix and then link the extender to the matrix by reaction with the activated groups introduced. Typical activation reagents are bifunctional in the sense that they are able to react twice with electrophilic functional groups. Illustrative examples are epihalohydrins, bisepoxides, CNBr etc. Other bifunctional reagents require an intermediary activation reaction, for instance allyl glycidyl ether and styryl glycidyl ether. For the latter reagents the first reaction takes place at the oxirane group whereafter activation is caused by addition of halogen (X2, OH or other compounds providing positive halogen) to the carbon-carbon double bond. The bifunctional reagents often give rise to a stable bridge between the extender and the base matrix, which in the preferred cases contains a straight or branched hydrocarbon chain that is broken by one or more oxygen atoms (ether structures) or amino nitrogens and optionally also substituted with one or more hydroxy or amino groups. The preferred bifunctional reagents should give rise to bridges that are stable against hydrolysis in the pH interval used in liquid chromatography, i.e. in most cases pH 3-12. This often means that the bridge should be devoid of ester groups and groups of similar or less hydrolytic stability, for instance the bridges should have at most one oxygen or nitrogen atom linked to one and the same carbon atom. Typically, the bridge has a length that is less than 30 atoms.
If properly utilized, the bifunctional reagent used for attaching the extender to the base matrix will cause no or a very low number of intra- and intermolecular crosslinks in the extender.
Affinity Structures/ligands
Affinity properties may be inherent in the structure of the extender or may be introduced by chemical coupling of the appropriate ligand structures/groups (affinity ligands) to the extender. This means that the extender often is substituted with one, in the preferred case two or more, ligands/groups that have affinity for the substance/substances intended to be adsorbed. Typical affinity ligands/groups are:
1. Positively charged groups (primary, secondary, tertiary or quaternary amine groups).
2. Negatively charged groups (for instance carboxy, phosphonic acid, sulphonic acid, etc.).
3. Amphoteric groups.
4. Groups having a specific affinity (for instance bioaffinity groups), such as between IgG-binding protein (Protein A, G, L, etc.) and IgG, lectin and carbohydrates, antigen/hapten and antibody, (strep) avidin and biotin.
5. Complementary nucleic acids/oligonucleotides).
6. Groups which exhibit xcfx80-electron systems.
7. Chelate groups.
8. Hydrophobic groups, etc.
With the aid of these affinity ligands/structures, the inventive method can be performed as affinity chromatography, such as ion-exchange chromatography, biospecific affinity chromatography, hydrophobic interaction chromatography, xe2x80x9creverse phase chromatographyxe2x80x9d, chelate chromatography, covalent chromatography, etc or corresponding batch mode adsorptions.
The ligand may be introduced before or after the extender has been attached to the surface of the base matrix. One way of doing this contemplates reacting the appropriate group of the extender, such as carboxy, amino, hydroxy, etc, with a suitable bifunctional reagent, such as CNBr, bisepoxide or corresponding epihalohydrin, allyl glycidyl ether etc, to introduce the desired reactivity which in turn is reacted with a compound that will introduce the affinity concerned. In case the compound exhibits a group reactive with a group on the extender no extra derivatization of the extender is required. The conditions and reagents should be selected so as to minimize cross-linking of the extender.
The introduction of the affinity ligand often contemplates inserting a linker between the ligand and the extender. Such linkers are often hydrophilic in the sense that they contain a straight, branched or cyclic hydrocarbon chain which may be broken by oxygen (ether) and/or nitrogen (amino) atoms and/or substituted with hydroxy and/or amino groups. The demands on the linker mostly is the same as on the bridge attaching the extender to the base matrix. See above.
The poor effect obtained with native protein A may be explained in terms of optimal ratios between the size of the extender and the affinity structure/group. Compare that native Protein A is much greater then the extender used in the experimental part (Example 11) (dextran Mw 10,000 Da). This also points to the fact that with the present knowledge it is believed that the largest effects with the invention will be achieved for smaller affinity groups that normally are found among groups 1, 2, 3, 6, 7 and 8 as defined above. Suggestive-wise the ratio between the Mws for the affinity structure and the naked extender should be less than 1, such as less than 0.1.
Base Matrices
The base matrix of the beads may be of organic or inorganic nature as known for beads used in chromatography on fluidized and packed beads. It may be porous or non-porous. It is often a polymer, such as glass, a synthetic polymer or a biopolymer. The base matrix may be a hydrophobic polymer, for instance a styrene-divinyl benzene copolymer, which has been hydrophilized on inner and/or outer surfaces by being coated with the appropriate hydrophilic polymer (often a polymer exhibiting hydroxy and/or amino groups) or by other means, for instance oxidized to introduce hydrophilic groups of the type given above. Alternatively, the base matrix may be a water-insoluble hydrophilic polymer, for instance agarose, cellulose, dextran starch, etc., which has been cross-linked to give the desired porosity and stability, if necessary. At the priority date, agarose was the polymer of choice, preferably in cross-linked form. For further discussion about selection of polymers in base matrices, see WO-A-9200799 (Kem-En-Tek A/S/Upfront Chromatography A/S), WO-A-9218237 (Pharmacia Biotech AB), PCT/SE96/01431 (Pharmacia Biotech AB) and WO-A-8603136 (Graves and Burns, University Patents Inc).
Density of Beads and Filler Materials
For upward fluidization, the density of the final beads (mean density, wet state) shall be higher than the density of the fluid in which the beads are to be used, i.e. for aqueous fluids  greater than 1 g/cm3, for instance xe2x89xa71.1 g/cm3, such as xe2x89xa71.2 g/cm3, (measured in the buffer used to maintain the bed in a fluidized state). For downward fluidization, the opposite applies, i.e. the density of the final beads (mean density, wet state) shall be lower than the density of the fluid in which the beads are to be used, i.e. for aqueous fluids  greater than 1 g/cm3. For stirred suspension the density of the beads is less critical.
The beads used in the inventive method may or may not comprise filler material in order to give the beads the appropriate density for the intended us. The filler material may be magnetic or non-magnetic.
Some bead matrix materials, such as porous glass beads and other porous inorganic materials, may have by themselves the appropriate density in the wet state and therefore do not need to contain a filler for controlling the density.
Organic polymers, on the other hand, often has a density close to the densities of the fluids used in the adsorption processes contemplated. In this case it is often of great advantages to incorporate filler particles as known in the art for fluid bed adsorptions, see WO-A-9200799 (Kem-En-Tek A/S/Upfront Chromatography A/S), WO-A-9218237 (Pharmacia Biotech AB) and PCT/SE96/01431 (Pharmacia Biotech AB). One, preferably two or more, particles are introduced per bead. Depending on direction of the flow for fluidization (downward or upward), the filler should have a density lower or higher, respectively, than the fluid to be used. For aqueous fluids this means  less than 1 g/cm3 (downward) or  greater than 1 g/cm3 (upward). Typical fillers are particles of glass, quartz and silica, metals, metal salts, metal alloys etc. See further WO-A-920799 (Kem-En-Tek A/S/Upfront Chromatography A/S) and WO-A-9218237 (Pharmacia Biotech AB). For flow rates  greater than 300-400 cm/h (upward direction), the density of the filler should be xe2x89xa73 g/cm3, with the preferred filler materials being heavy metals.
The particle size of the filler material is normally within the range of 1-200 xcexcm, with the provision that it always is less than the beads. For filler materials to be used in porous beads and having dens Vies xe2x89xa73 g/cm3, typical particle sizes are 1-70 xcexcm, with a preference to a range of 15-50 xcexcm.
For porous beads, the geometrical shape of the filler particles is important. Preferred shapes include spheres, ellipsoids, droplets, noodle shapes, bean shapes and aggregates/agglomerate of this forms of particles. A particular preference is given to continuously rounded shapes. See further PCT/SE96/01431 (Pharmacia Biotech AB). This becomes particularly important in case the pore sizes permit penetration of the substance(s) to be adsorbed.
The filler content of the beads is determined by factors such as the desired density and capacity of the final beads, flow rates to be used, type and density of filler material, ligand, substance to be adsorbed etc. Typical optimal filler contents are to be found in the range 1-100% (w/w, wet beads).
Bead Size
The population of beads used in each particular application may be monodisperse or contains beads covering a certain size range with a certain bead size distribution and mean bead size. Suitable mean bead sizes and size ranges are typically found within the range of 10-1,000 xcexcm, with preference to a range of 50-700 xcexcm. In many cases of fluidized beds (expanded beds), the lower limit is determined by the fact that the beads shall not be able to escape through the outlet or the inlet of the vessel used for fluidizing. Other factors which influence the choice of bead sizes include the desired capacity, the ligand concerned, the specific substance or substances to be adsorbed from the sample etc.
The ratio between the total surface area of the beads (inner plus outer surfaces) and the total bead volume is highly significant to breakthrough capacity. Larger relative contact surface areas (small beads) lead to a higher breakthrough capacity. The total capacity, on the other hand, is only marginally affected.
For expanded beds stabilized by a flow evenly distributed across the cross-sectional area of the bed, typical bead size distributions are such that 95% of the beads fall within a range whose width is 0.1 to 10 times the mean bead diameter, preferably 0.3 to 3 times the mean bead diameter. The exact particle size distribution to be selected will depend on Factors, such as flow rate, mean bead diameter, density of beads, density of fluid etc. A too wide particle size distribution will result in an increased risk for elutriation and/or sedimenting of large proportions of beads. The size distribution may be unsymmetrical, for instance the proportion of beads in the lower part of a range can be larger than the proportion in the upper part. Although less preferred with present production technology, a future alternative to size distribution in this particular application is to have a distribution in the densities of the beads used. Bead populations in which both the size and the density of the individual beads vary can also be used.
For magnetically stabilized fluidized beds or turbulent fluidized beds and stirred suspensions, the demand for a size and/or a density distribution within the bead population used is less critical. In these cases also monodisperse bead populations may be used.
Porosity of Beads
It is preferred that the final beads are porous with open pores allowing the substance to be adsorbed to penetrate the internal of the beads. Optimal porosity can be determined from the size of the substance or substances to be adsorbed. It appears as if the extender in some unknown way would facilitate transportation of substances within the beads. It is therefore believed that the range for acceptable Kav-values are broader than for beads without extenders, i.e. 0.1-0.95 compared to 0.40-0.95 without extenders. For a definition of Kav see L. Hagel in xe2x80x9cProtein Purification, Principles, High Resolution, and Applicationsxe2x80x9d, J-C Janson and L Rydxc3xa9n (Eds), VCH Publishers Inc. New York, 1989, p. 99.
Samples to be Applied
The samples to be applied in the inventive method may be of the same type as those earlier applied in chromatographic processes on packed and non-packed beds, or in batch adsorption processes on turbulent fluidized beds and agitated suspensions. The inventive beads and methods can be applied to the treatment of processed and unprocessed supernasants/culture media from fermentors and other cell culture vessels, serum, plasma, beverages etc.
Either the adsorbed substance or the sample is further processed.
The invention will function for the separation of compounds of various molecular weights and types. Examples are macromolecules, e.g. with molecular weights xe2x89xa75,000 Dalton, such as polysaccharides, proteins/polypeptides and nucleic acids and synthetic water-soluble polymers. Also substances with molecular weight xe2x89xa65,000 Daltons may be adsorbed according to the invention. There is normally no upper limit in molecular weight, even though the process is normally limited to the adsorption/separation of compounds that have a molecular weight below 1,000,000.
Miscellaneous Aspects of the Manufacture of Beads
The manufacture of the base matrix beads, introduction of binding groups, addition of filler, etc., are effected in a known manner, while ensuring that the beads will be suited for adsorption purposes in accordance with the aforegoing. The beads are sieved, when necessary, to obtain a suitable size fraction.
A Second Aspect of the Invention
A second aspect of the invention, is a population of beads containing filler material (bead fraction) according to the above, which is suitable for use as a matrix in adsorption/separation processes, particularly chromatographic processes, effected on expanded/fluidized beds in accordance with the aforegoing. This aspect of the invention also includes the population in the form of a stable expanded bed according to the above, placed in a column with openings for inlet and outlet of a fluidizing flowing liquid. This second aspect includes bead populations which lack affinity groups, e.g. preactivated forms. As discussed above the beads in the population may cover a certain size and/or density range or be homogeneous with respect to these features (monodisperse).
The following experimental part discloses the manufacture of the bead population most preferred at the priority filing of this application.
Synthesis